OspA, an outer surface protein of the spirochete B. burgdorferi, the etiologic agent of Lyme disease, is a vaccine candidate of proven efficacy in rodents. Variants of B.burgdorferi that are not susceptible to killing with antibody to Osp's in vitro have been identified in several laboratories and the concern has been raised that such variants may significantly diminish efficacy of vaccines based on Osp antigens. A polyclonal monospecific anti-OspA antiserum obtained from rhesus monkeys that had been immunized with pure lipidated OspA was heat-inactivated and used to select antibody-resistant variants of the HB19 strain of B. burgdorferi in vitro. Variants were cloned twice in solid medium and characterized by SDS- PAGE and Western blot, using antibodies specific for OspA (polyclonal) and OspB (monoclonals), and by PCR. The most frequently appearing phenotypes were deletion mutants expressing the N-terminus of OspA fused to the C-terminus of OspB. Clones of four of these phenotypes were assessed for their ability to resist antibody-dependent, complement-mediated killing (ADCK) in vitro, using the same anti-OspA polyclonal antiserum used for the selection of the mutants, and rhesus complement. Surprisingly, all of the variants that were resistant to antibody-mediated killing (AMK) were susceptible to ADCK. However, the antibody concentration necessary to achieve 50 % killing (ADCK50) was between 1 and 100-fold higher than the ADCK50 of the HB19 wild-type strain. Susceptibility to ADCK depended on the mutant phenotype. We are currently analyzing this correlation, to understand which portions/ epitopes of the OspA and OspB molecules are targeted by the ADCK and the AMK mechanisms. OspB is targeted in ADCK by anti-OspA antibodies, as indicated by a 10-fold increase of the ADCK50 of the wild-type strain when the anti-OspA antiserum is pre-incubated with an excess of recombinant OspB. This could explain the ADCK of mutants that have lost the C-terminus of OspA, which contains the neutralizing epitopes, but retain the C-terminus of OspB. A dual mode of protection by anti- OspA antibody-mediated vaccines has been postulated, one acting on the spirochetes within the tick prior to transmission, and the second upon delivery within the host skin. Since it is likely that the tick blood-meal may be depleted of complement by the action of de-complementing factors within tick saliva, killing of spirochetes within the tick may depend exclusively on antibody, whereas ADCK may be the prevailing mechanism in the skin. Our results suggest that escape mutants may be able to avoid AMK within the tick, but not ADCK within the host skin. If, however, the expression of OspA is down-regulated immediately prior to transmission, as recent experiments indicate, then OspA mutants such as the ones we have isolated will readily escape the immunity elicited by OspA vaccines.